1. Field of the Invention
The present invention relates to an enzyme electrode and more particularly an enzyme electrode containing an enzyme and a coenzyme as immobilized.
2. Description of the Prior Art
In order to determine concentration of a substance in a solution, it has been proposed to use an enzyme electrode comprising an enzyme, which is specifically reactive with the substance, that is, a substrate of the enzyme, and is immobilized on the surface of the electrode. According to its principle, the electrode is immersed in the solution to generate an enzyme reaction on the electrode surface and the amount of the reaction product is electrochemically detected, thereby to determine the concentration of the substance in the solution. Up to the present, enzyme electrodes using oxidation enzymes such as glucose oxidase have been proposed and produced.
On the other hand, some of oxidoreductases require existence of coenzymes. For example, dehydrogenases such as alcohol dehydrogenase and lactate dehydrogenase, which are industrially very useful, require existence of nicotinamide adenine dinucleotide (NAD) as a coenzyme. When these enzymes are applied to an enzyme electrode, it is necessary to detect a coenzyme derivative produced by the enzyme reaction, for example, reduced type NAD (NADH) in the case of the alcohol dehydrogenase, by the electrode to determine the amount of the substrate.
Heretofore, in most of such enzyme electrodes, only enzymes have been immobilized on the electrodes, and the coenzymes have been supplied from the solution without immobilization. For example, "Analytica Chimica Acta", 214 (1988), pp 161-172, discloses a report on an ethanol measurement system by a flow injection analysis method by means of a coenzyme solution. On the other hand, it has been proposed to immobilize an enzyme and a coenzyme at the same time on an electrode, by applying a coating of a solution containing an enzyme and a coenzyme on a carbon electrode, as described in the Japanese Patent Application Number 6961/1981.
Because a coenzyme solution is not stable enough to permit long time stock and is very expensive, it is not advantageous to supply the coenzyme from the solution. Further, the flow injection analysis method requires a large scale apparatus and so is not convenient for a case where simple measurement is desired. For these reasons, it is desirable to use an electrode on which both of an enzyme and a coenzyme are immobilized. The known electrode, on which the enzyme and the coenzyme are simply coated as above mentioned, however, has defects of low sensitivity owing to the fact that the coenzyme cannot be immobilized in a sufficiently large amount, and insufficient durability for repeated use owing to the fact that the coenzyme has strong tendency of dissipation into the solution because of its small size of molecule.